JPH08173169A - Regulatory factor for manifesting nitrilase gene and the gene - Google Patents

Abstract

PURPOSE: To obtain a regulatory factor having nitrilase gene promoter-activating action, composed of plural kinds of peptide, capable of producing nitrilase through coexistence with a nitrilase gene in Rhodococcus microbes, and to obtain the gene coding this factor. CONSTITUTION: This new regulatory factor has action to activate a nitrilase gene promoter, being composed of a polypeptide having an amino acid sequence of formula I and a second polypeptide having an amino acid sequence of formula II. The other objective new gene codes for this regulatory factor. Coexistence of this gene with a nitrilase gene containing a promoter domain in Rhodococcus microbes enables nitrilase production. The regulatory factor and the gene are obtained by the following process: chromosome DNAs are separated from a Rhodococcus erythropolis SK92 strain, a DNA library is prepared by using these DNAs, and then screened to take the gene coding for the regulatory factor, and the gene is then manifested to obtain the regulatory factor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to regulatory factors involved in the expression of nitrilase gene and gene DNA encoding them. Specifically, a regulatory factor derived from Rhodococcus erythropolis SK92 strain and having an action of activating the nitrilase gene promoter, and a gene DN encoding them
The present invention relates to a recombinant plasmid containing A and these gene DNAs, and a transformant transformed with the recombinant plasmid.

[0002]

BACKGROUND OF THE INVENTION Chemical synthetic methods and biological methods are known as methods for producing corresponding organic acids from nitrile compounds. In the biological method, since a microorganism or an enzyme derived from the microorganism is used as a hydrolysis catalyst for a nitrile compound, there is an advantage that an organic acid can be produced under mild conditions. Microorganisms belonging to the genus Rhodococcus are known as microbial catalysts for hydrating or hydrolyzing nitriles to produce the corresponding amides or organic acids (JP-A-3-251192).
JP-A-62-91189, JP-A-2-470 and JP-A-2-8419
(See each gazette of No. 8).

In the hydrolysis of nitrile by a nitrilase gene cloned by a gene recombination method, a large number of copies of the gene can be present in the cells.
It can be expected that the catalytic ability of microorganisms will be dramatically increased as compared with conventional methods. The present inventors cloned the nitrilase gene of Rhodococcus erythropolis SK92 strain to prepare a plasmid having this gene inserted downstream of the Escherichia coli lactose promoter in order to prepare a microbial catalyst having higher catalytic activity. Escherichia coli introduced with this plasmid exhibited high nitrilase activity when cultured in the presence of IPTG (isopropyl-β-D-thiogalactoside). Furthermore, when used as a microbial catalyst, a recombinant Rhodococcus bacterium with better functionality was produced. That is, the nitrilase gene is used as a Rhodococcus-E.
No. 64589 and No. 5-68566), and introduced into Rhodococcus. However, nitrilase activity is not expressed, and a method for expressing it in a recombinant Rhodococcus bacterium has been desired.

[0004]

[Means for Solving the Problems] The present inventors have found that no expression is observed in the genus Rhodococcus because the promoter of the nitrilase gene is not functioning, and a gene encoding a regulatory factor required for the function of the promoter is expressed. Is S
We thought that it might exist somewhere on the K92 strain chromosomal DNA. As a result of the search, its presence was found upstream of the nitrilase structural gene, and the nitrilase activity was expressed in the recombinant bacteria of the genus Rhodococcus, resulting in the completion of the present invention.

That is, the present invention has a function of activating the nitrilase gene promoter and is composed of two components, a polypeptide having the amino acid sequence of SEQ ID NO: 1 and a polypeptide having the amino acid sequence of SEQ ID NO: 2. Factors and the genetic DNAs that encode them.

The present invention will be described in detail below. The present invention is carried out by the following steps. (1) Preparation of SK92 strain chromosomal DNA Chromosomal DNA is isolated and prepared from Rhodococcus erythropolis SK92 strain.

(2) Preparation of DNA library After cutting the chromosomal DNA with a restriction enzyme, a DNA fragment fraction containing the target gene is detected by Southern hybridization using the SK92 strain nitrilase gene as a probe. This fraction is used as a library by inserting it into a complex plasmid vector capable of replicative growth in cells of Escherichia coli and Rhodococcus.

(3) Preparation of Escherichia coli Transformant and Selection of Recombinant DNA Escherichia coli transformant is prepared from the recombinant library prepared in step (2), and prepared in step (2) Transformants containing the target recombinant DNA are selected by the colony hybridization method using the probe thus prepared.

(4) Preparation of recombinant plasmid A plasmid is prepared from the recombinant prepared in step (3).

(5) Transformation of bacteria of the genus Rhodococcus and nitrilase activity of transformants Bacteria of the genus Rhodococcus are transformed with the obtained plasmid, and the nitrilase activity is measured.

(6) Deletion plasmid and nitrilase activity A plasmid is prepared by removing various regions from the plasmid obtained in step (4), and a region other than the nitrilase structural gene region, which is essential for expression, is specified. The plasmid prepared here does not necessarily have to be capable of replicating and propagating in E. coli cells, and may contain a DNA region capable of replicating and propagating in bacterial cells belonging to the genus Rhodococcus.

(7) Determination of base sequence The base sequence of the region specified in step (6) is determined.

As the composite plasmid vector,
pK1, pK2, pK3 and pK4. These plasmids are Rhodococcus rhodochrous AT
Introduced in CC 12674, R. rhodochrous ATCC 1267
4 / pK1 (FERM BP-3728), R. rhodochrous
ATCC 12674 / pK2 (FERM BP-3729), R. r
hodochrous ATCC 12674 / pK3 (FERM BP-373
0) and R. rhodochrous ATCC 12674 / pK4 (FERM
BP-3731) has been deposited at the Institute of Biotechnology, Institute of Industrial Science and Technology (see Japanese Patent Laid-Open No. 5-68556).

The DNA region capable of replicative growth in bacterial cells belonging to the genus Rhodococcus includes plasmid pR
C001, pRC002, pRC003 and pRC0
Those derived from a plasmid selected from 04 are used, and these may be the entire plasmid or one fragment. The above plasmids are Rhodococcus rhodochrous ATCC 4276 and ATCC 1434, respectively.
9, ATCC 14348 and IFO 3338 strains (see JP-A-5-68556).

Rhodococcus erythropolis SK92
The strain is FERM BP-3324 and also the plasmid pSK1 containing the nitrilase gene and the regulatory gene.
08 is a transformant containing this JM109 / pS
Deposited as K108 (FERM P-14569) at the Institute of Biotechnology, Institute of Biotechnology, AIST. SK92
The strain was identified as a genus Rhodococcus based on its mycological properties (see Japanese Patent Application Laid-Open No. 3-280889), and was further identified as Rhodococcus erythropolis based on the following detailed mycological properties.

Test item Test result Adenine decomposition + Tyrosine decomposition + Urea decomposition + Assimilating inositol + Maltose-mannitol + Rhamnose-sorbitol + m-hydroxy-sodium benzoate-Sodium benzoate + Sodium citrate + Sodium lactate + Testosterone + Acetamide + sodium pyruvate + 0.02% Growth in the presence of sodium azide + Growth at 10 ° C + Growth at 40 ° C-0.001% Growth in the presence of crystal violet-0.3% Growth in the presence of phenylethanol-5 Growth in the presence of% NaCl + 7 Growth in the presence of 7% NaCl +

A detailed description will be given below with reference to embodiments. However, the present invention is not limited to these examples. As a reference example, the cloning of the SK92 strain nitrilase gene and the expression in Escherichia coli and Rhodococcus bacteria are also specified.

[0018]

【Example】

(1) Preparation of SK92 strain chromosomal DNA The SK92 strain was shake-cultured in 100 ml of MY medium (0.5% polypeptone, 0.3% bacto yeast extract, 0.3% bacto malt extract) at 30 ° C. for 72 hours. The cells were collected afterwards and the cells were diluted with a saline-EDTA solution (0.
1M EDTA, 0.15M NaCl (pH8.0) 4m
The suspension was suspended in 1 l, 8 mg of lysozyme was added, and the mixture was shaken at 37 ° C for 1 to 2 hours and then frozen. Next, 10 ml Tris-S
DS solution (1% SDS, 0.1M NaCl, 0.1MTr
Is (pH 9.0) was added with gentle shaking, and further proteinase K (Merck) (final concentration 0.1 mg).
Was added, and the mixture was shaken at 37 ° C for 1 hour, and further shaken at 60 ° C. Next, an equal amount of TE-saturated phenol was added and after stirring (T
E: 10 mM Tris, 1 mM EDTA (pH 8.
0)) Centrifuge, take the upper layer, add twice the amount of ethanol, and wind the DNA with a glass rod to obtain 90%, 80%, 7%
Phenol was sequentially removed with 0% ethanol. Next, the DNA was dissolved in 3 ml of TE buffer, a ribonuclease A solution (100 ° C., 15 minutes heat-treated) was added to 10 μg / ml, and the mixture was shaken at 37 ° C. for 30 minutes. After further adding proteinase K and shaking at 37 ° C. for 30 minutes, an equal amount of TE-saturated phenol was added and centrifuged to separate the upper layer and the lower layer. After repeating this operation twice for the upper layer, the same amount of chloroform (containing 4% isoamyl alcohol) was added and the same extraction operation was repeated (hereinafter,
This operation is called phenol treatment). Then 2 on the upper layer
A double amount of ethanol was added, and the DNA was wound up with a glass rod and collected to obtain a chromosomal DNA sample.

(2) Preparation of DNA library A DNA fragment containing the SK92 strain nitrilase gene is pU
Plasmid pSK00 integrated into C118 vector
After cutting 2 (see Reference Example) with the restriction enzyme SalI, 1.1
The kb SalI fragment was separated by 0.7% agarose electrophoresis, cut out from the gel and collected. Cleavage with a restriction enzyme was performed as follows. 10 times the concentration of the restriction enzyme buffer 10 μl against pSK00210 μl, sterilized water 78 μl
1 and 2 μl of the restriction enzyme SalI were added and reacted at 37 ° C. for 2 hours. Agarose gel electrophoresis was performed on a standard sample obtained by cleaving the SK92 strain chromosomal DNA with EcoRI.
The 1.1 kb SalI fragment was labeled using the DIG DNA Labeling Kit (Boehringer Mannheim Co., Ltd.), and this was used as a probe for Southern hybridization (So.
About 14k by uthern EM, Mol.Bionl.98 503 (1975))
The DNA fragment of b was detected. A DNA fragment fraction containing a 14 kb fragment that hybridized with the probe was cut out from an agarose gel and cut with EcoRI pK4 [FERM BP-37311: Rhodococcus genus plasmid pRC004 and E. coli vector pH.
It was inserted into a combination of SG299 (see JP-A-5-64589 and JP-A-5-68566). The pK4 fragment used for the vector was prepared as follows. pK 410 μl, 10 times concentration limiting enzyme buffer 10 μl, sterilized water 77 μ
1, 2 μl of restriction enzyme EcoRI were added, and the mixture was reacted at 37 ° C. for 2 hours, treated with phenol, precipitated with ethanol, dried, and dissolved in 50 μl of sterilized water. Further, 1 μl of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 μl of 10 times concentration buffer and 39 μl of sterilized water were added, and after reaction at 65 ° C., phenol treatment and ethanol precipitation were performed, followed by drying and dissolving in sterilized water. 1 μl of DNA fragment fraction containing 14 kb fragment
Was reacted with EcoRI-cut pK4 prepared as described above and a ligation kit (Takara Shuzo Co., Ltd.) at 4 ° C. overnight to insert into pK4, thereby
It was a library.

(3) Preparation of transformant and recombinant DN
Selection of A. Escherichia coli JM109 strain was mixed with LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaC).
l) 1 ml was inoculated and precultured at 37 ° C. for 5 hours, and 100 μl of this culture was added to SOB medium (2% bactotryptone,
0.5% Bacto yeast extract, 10 mM NaCl,
2.5 mM KCl, 1 mM MgSO ₄ , 1 mM MgCl
₂ ) It was added to 50 ml and cultured at 18 ° C. for 20 hours. After collecting the cells by centrifugation, cold 13 ml TF solution (20 mMPI
PES-KOH (pH 6.0), 200 mM KCl, 1
13 ml of 0 mM CaCl ₂ and 40 mM MnCl ₂ ) was added, and the mixture was left standing at 0 ° C. for 10 minutes and then centrifuged again. After removing the supernatant, the precipitated Escherichia coli was suspended in 3.2 ml of cold TF solution, 0.22 ml of dimethylsulfoxide was added, and the suspension was added at 0 ° C for 10 minutes.
Let stand for a minute. Competent cell 20 produced in this way
10 μl of the solution (DNA library) containing the recombinant plasmid prepared in step (2) was added to 0 μl,
After 30 minutes of standing at 40 ° C., heat shock at 42 ° C. for 30 seconds and cooling at 0 ° C. for 2 minutes, then SOC medium (2% bactotryptone, 0.5% bacto yeast extract, 20 mM glucose, 10 mM NaCl, 2.5 mM KCl, 1 mMM
Add 0.8 ml of gSO ₄ , 1 mM MgCl ₂ ) at 37 ° C
The cells were cultivated with shaking at 60 minutes. 200 μl of each is spread on LB agar medium containing 100 μg / ml of ampicillin,
Cultured at 37 ° C. From the transformant colonies grown on the agar medium, transformants having the nitrilase gene were selected by the colony hybridization method. That is, the transformant grown on the agar medium was transferred onto a nylon membrane (Biodyne A: Nippon Pole Co., Ltd.), the cells were dissolved to fix the DNA, and then this step was performed.
Treated with the probe (1.1 kb fragment) prepared in (2),
Using the DIG Luminescent Detection Kit (Boehringer Mannheim Co., Ltd.), colonies containing the target recombinant DNA were selected.

(4) Preparation of Recombinant Plasmid The transformant selected in step (3) was cultured in 100 ml of LB medium at 37 ° C. overnight, and the cells were collected and washed with sterile water to prepare solution I ( 2 mM glucose, 10 mM EDTA, 2
5 ml of 5 mM Tris.HCl (pH 8.0) and 25 mg of lysozyme were added, and the mixture was left at 0 ° C. for 30 minutes. solution
II (1N NaOH, 5% SDS) (10 ml) was added, and the mixture was allowed to stand at 0 ° C. for 5 minutes to give a solution III (3M sodium acetate (pH
7.5 ml of 4.8) was added and the mixture was left at 0 ° C. for 30 minutes. This was centrifuged, 50 ml of ethanol was added to the supernatant, and the supernatant was removed by centrifugation and 5 ml of solution IV (10
mM sodium acetate, 50 mM Tris / HCl (pH
8.0) and ribonuclease A solution (10 mg / ml)
Was added in an amount of 2.5 μl and left at room temperature for 20 minutes. 12 to this
After adding ml of ethanol, the mixture was centrifuged, dried, and dissolved in sterile water.

(5) Transformation of bacteria of the genus Rhodococcus and nitrilase activity of transformants Cells in the logarithmic growth phase of Rhodococcus rhodochrous ATCC 12674 strain were collected by centrifugation, washed with ice-cold sterile water three times, Suspended in sterile water. 10 μl of the bacterial cell suspension was mixed with 1 μl of the plasmid pSK104 of step (4) and cooled on ice. Put a mixture of DNA and bacterial cells in the chamber, and introduce the gene transfer device CET-200 type (JASCO)
The electric pulse treatment was performed with an electric field strength of 3.8 kV / cm, a pulse width of 1 ms, and a pulse count of 20. The electric pulse treatment solution was allowed to stand for 10 minutes under ice cooling and heat shocked at 37 ° C. for 10 minutes to prepare MYK medium (0.5% polypeptone, 0.3% bacto malt extract, 0.3% bacto yeast extract, 0%). 0.2% KH ₂ PO ₄ , 0.2% K ₂ HP
After adding 500 μl of O ₄ (pH 7.0) and shaking culture at 26 ° C. for 3 hours, M containing 75 μg / ml kanamycin
It was applied to YK agar medium and cultured at 26 ° C. for 3 days. The recombinant Rhodococcus bacterium thus prepared was inoculated into 10 ml of MYK medium (containing 50 μg / ml kanamycin), and 3
It precultured at 0 degreeC for 24 hours. 1 ml of this culture is GGP
Medium (1.5% glucose, 0.1% bacto yeast extract, 1.0% sodium glutamate, 0.05% K
H ₂ PO ₄ , 0.05% K ₂ HPO ₄ , 0.05% Mg
_{SO 4 · 7H 2 O (pH7.2} )) 100ml (75μ
(containing g / ml kanamycin) and ethylene cyanohydrin (ECH) 1.5% as an inducer, 30 ° C
The cells were cultured for 48 hours. After collecting the cells, the cells were suspended in a 50 mM phosphate buffer solution (pH 7.7), and a part of the cells was suspended for 100 m.
In the same buffer containing M acrylonitrile at 30 ° C., 2
The reaction was allowed for 0 minutes. The reaction was stopped by adding 1N hydrochloric acid, and the produced acrylic acid in the reaction solution was measured by high performance liquid chromatography. As a result, in the recombinant Rhodococcus ATCC 12674 / pSK104, 8
Generation of mM acrylic acid was observed. By this,
It was revealed that a gene encoding a regulatory factor required for nitrilase expression exists upstream or downstream of the nitrilase structural gene.

(6) Deletion plasmid and nitrilase activity Since it was considered that a lot of regions not required for nitrilase expression still remained in pSK104, various deletion plasmids were prepared and the nitrilase activity was measured. (Table 1,
(Fig. 1)

[0024]

As a result, ATCC12674 / pSK1
08 (6.2 kb HindIII-EcoRV fragment) (FIG. 2) showed high nitrilase activity. Furthermore, as a result of constructing a deletion plasmid and searching for the position, it was found that the gene encoding the regulatory factor was located in the region fairly upstream of the nitrilase structural gene (about 3 kb BamHI-EcoRV fragment).

(7) Determination of nucleotide sequence The nucleotide sequence of the gene encoding the regulatory factor essential for the expression of nitrilase revealed in step (6) was determined using Pharmacia Fluorescence Sequencer ALFI I. As a result, the base sequence shown in SEQ ID NO: 5 was obtained, and two open reading frames having the amino acid sequences shown in SEQ ID NOs: 1 and 2 were found. Amino acid sequence database NBRF (National Biomedical Rese
By comparison with arch fo-undation), it was estimated that the regulatory factor belongs to the family of two-component regulatory system. The nucleotide sequences of these open reading frames are shown in SEQ ID NOs: 3 and 4.

Reference Example (1) Preparation of SK92 strain chromosomal DNA In the same manner as in Step (1) of Example, SK92 strain chromosomal DNA was prepared to obtain a chromosomal DNA preparation.

(2) Preparation of probe and preparation of DNA library 10 μl (1/20 dilution) of substrate DNA, 10 μl of 10-fold concentration reaction buffer, 4 μl of 5 mM dNTP, 5′-AACTGCTGGGA (AG) CACTTCCA-3 as primer # 1 '(2
0 nucleotides, corresponding amino acid sequence NCWEHF
Q) and 5'-GA (AG) TA (AG) TG (A
G) CC (CG) AC (ACTG) GG (AG) TC-3 ′ (20 nucleotides,
Corresponding amino acid sequence DPVGHYS 5 μl each (50
1 μl of Tth DNA polymerase (Toyobo Co., Ltd.) was added to make 100 μl. All the primers were prepared on the basis of amino acid sequences having high homology with various known nitrilases. Reaction is 93 ° C, 30 seconds (denaturation step), 45
The reaction was terminated by carrying out 50 cycles of incubation at 30 ° C. for 30 seconds (annealing step), 72 ° C. for 2 minutes (extension step). A 410 bp DNA fragment encoding the SK92 strain nitrilase gene was obtained from the obtained reaction solution. The DNA fragment thus obtained was labeled with DIG DNA Labeli
It was labeled with ng Kit (Boehringer Mannheim Co., Ltd.) and used as a probe. SK92 strain chromosomal DNA50
10 μl of 10 times concentration limiting enzyme buffer per μl, sterilized water 37
μl and 3 μl of the restriction enzyme SalI were added and reacted at 37 ° C. for 2 hours, followed by ethanol precipitation and agarose electrophoresis for separation. And the DNA around 1.1 kb
The fragment was recovered using DNAPREP (Diatron Co., Ltd.). This DNA fragment was transformed into E. coli vector pUC11 using a ligation kit (Takara Shuzo).
8 was inserted into the SalI site to prepare a recombinant DNA library. The pUC118 fragment used for ligation was prepared as follows. pUC118 10 μl to 10 μl concentration limiting enzyme buffer 10 μl, sterilized water 77 μl,
After adding 2 μl of restriction enzyme SalI and reacting at 37 ° C. for 2 hours,
Phenol-treated, ethanol-precipitated and dried to 50
Dissolved in μl sterile water. Furthermore, 1 μl of alkaline phosphatase (Takara Shuzo Co., Ltd.), 10 times concentrated buffer solution 1
0 μl and 39 μl of sterilized water were added and reacted at 65 ° C., treated with phenol, precipitated with ethanol, dried and dissolved in sterilized water.

(3) Preparation of transformant and recombinant DNA
Selection of competent cells of Escherichia coli JM109 strain were prepared in the same manner as in the step (3) of the example. To 200 μl of this competent cell, 10 μl of the solution containing the recombinant plasmid prepared in step (2) (DNA library) was added, and after leaving it at 0 ° C. for 30 minutes, heat shock at 42 ° C. for 30 seconds and at 0 ° C. After cooling for 2 minutes, 0.8 ml of SOC medium was added and cultivated with shaking at 37 ° C for 60 minutes. 200 μl of this was spread on LB agar medium containing 100 μg / ml of ampicillin, and 37
Cultured at ° C. From the transformant colonies grown on the agar medium, transformants having the nitrilase gene were selected by the colony hybridization method. That is, the transformant grown on the agar medium was transferred onto a nylon membrane (Biodyne A: Pall), the cells were dissolved to fix the DNA, and then the probe (410 bp fragment) prepared in step (2) was used. ) With DIG Luminescen
Using the t Detection Kit (Boehringer Mannheim Co., Ltd.), a colony containing the target recombinant DNA was selected.

(4) Preparation of Recombinant Plasmid and Construction of Restriction Enzyme Map The transformant selected in step (3) was prepared in the same manner as in Example step (4).
The preparation was done. Recombinant plasmid p thus obtained
SK002 was cleaved with several kinds of restriction enzymes to prepare a restriction enzyme map.

(5) Nitrilase Production Using Recombinant Escherichia coli and Conversion of Nitriles to Acids JM109 / pSK002 strain was mixed with 2 × YT medium (1.6%).
Bactrypton, 1.0% Bacto yeast extract,
0.5% NaCl) (containing 50 μg / ml ampicillin) 1
1 ml of this culture
1 was added to 100 ml of 2 × YT medium (containing 50 μg / ml ampicillin and 1 mM IPTG) and cultured at 37 ° C. for 14 hours. After collecting the cells, the cells were mixed with 50 mM phosphate buffer (p
H7.7), and a part thereof was reacted in the same buffer containing 100 mM acrylonitrile at 30 ° C. for 20 minutes. The reaction was stopped by adding 1N hydrochloric acid, and the produced acrylic acid in the reaction solution was measured by high performance liquid chromatography. As a control test, only JM109 strain was used. As a result, acrylic acid was not detected in the host JM109 strain, but the transformant JM109 / pSK0
In 02, the production of 18 mM acrylic acid was observed.

(6) Insertion of DNA Fragment Containing Nitrilase Gene into Composite Plasmid Vector SK92 strain nitrilase gene obtained in step (4) and a DNA fragment containing a region considered to be its promoter (5.8 kb BglII-HindIII) (Fragment) was excised from the chromosomal DNA and inserted into the composite plasmid vector pK4 to prepare plasmid pSK120.

(7) Transformation of bacteria of the genus Rhodococcus and nitrilase activity of transformants Cells in the logarithmic growth phase of Rhodococcus rhodochrous ATCC 12674 strain were collected by centrifugation, washed with ice-cold sterile water three times, Suspended in sterile water. 1 μl of the plasmid pSK120 of step (6) and 10 μl of the cell suspension were mixed and cooled on ice. Put a mixture of DNA and bacterial cells in the chamber, and introduce the gene transfer device CET-200 type (JASCO)
The electric pulse treatment was performed with an electric field strength of 3.8 kV / cm, a pulse width of 1 ms, and a pulse count of 20. The electric pulse treatment solution was allowed to stand under ice cooling for 10 minutes, heat shocked at 37 ° C. for 10 minutes, and 500 μl of MYK medium was added, and 2
Shake at 6 ° C. for 3 hours. It was applied to MYK agar medium containing 75 μg / ml kanamycin and cultured at 26 ° C. for 3 days. The Rhodococcus recombinant thus prepared was inoculated into 10 ml of MYK medium (containing 50 μg / ml kanamycin) and 3
Pre-incubate at 0 ° C. for 24 hours, add 1 ml of this culture to 100 ml of GGP medium (containing 75 μg / ml kanamycin),
ECH1.5% was added as an inducer and the mixture was heated at 30 ° C for 48 hours.
Cultured for hours. After collecting the cells, the cells were suspended in a 50 mM phosphate buffer solution (pH 7.7), and the nitrilase activity was examined as described in step (5). No activity was observed.

[0034]

[Effect] By coexisting a gene encoding this regulatory factor with a nitrilase gene containing a promoter region in a Rhodococcus bacterium, nitrilase can be produced.

[0035]

[Sequence list]

SEQ ID NO: 1 Sequence length: 244 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Rhodococcus erythropolis
erythropolis) strain name: SK92 sequence: 15 MetAlaGlyAlaAspValHisAlaGlnGlyGlyThrAsnArgArg 30 AlaArgIleLeuValValAspAspGluLysHisValArgThrMet 45 ValThrTrpGlnLeuGluSerGluAsnPheAspValValAlaAla 60 AlaAspGlyAspAlaAlaLeuArgGlnValThrGluSerAlaPro 75 AspLeuMetValLeuAspLeuSerLeuProGlyLysGlyGlyLeu 90 GluValLeuAlaThrValArgArgThrAspAlaLeuProIleVal 105 ValLeuThrAlaArgArgAspGluThrGluArgIleValAlaLeu 120 AspLeuGlyAlaAspAspTyrValIleLysProPheSerProArg 135 GluLeuAlaAlaArgIleArgAlaValLeuArgArgThrThrAla 150 GluProProHisGluAlaAlaValGlnArgPheGlyAspLeuGlu 165 IleAspThrAlaAlaArgGluValArgLeuHisGlyIleProLeu 180 GluPheThrThrLysGluPheAspLeuLeuAlaTyrMetAlaAla 195 SerProMetGlnValPheSerArgArgArgLeuLeuLeuGluVal 210 TrpArgSerSerProAspTrpGlnGlnAspAlaThrValThrGlu 225 HisValHisArgIleArgArgLysIleGluGluAspProThrLys 240 ProThrIleLeuGlnThrValArgGlyAlaGlyTyrArgPheAsp 244 GlyGluArgAla

SEQ ID NO: 2 Sequence length: 534 Sequence type: Amino acid Topology: Linear Sequence type: Protein Origin organism name: Rhodococcus erythropolis
erythropolis) strain name: SK92 sequence: 15 MetMetThrAspThrLeuProSerSerSerArgTrpThrLeuGlu 30 GlyProHisLeuGlnProLeuGlnGlyGluAlaLeuAlaAspLeu 45 HisAlaArgThrLeuGluMetIleThrSerGlyArgGluLeuHis 60 GluThrLeuGluValValAlaArgGlyIleGluGluLeuMetPro 75 GlyLysArgCysAlaIleLeuLeuLeuAspAsnThrGlyProVal 90 LeuArgCysGlyAlaAlaProThrMetSerAlaProTrpArgArg 105 TrpIleAspSerLeuValProGlyProMetSerGlyGlyCysGly 120 ThrAlaValHisLeuGlyGluProValIleSerTyrAspValAla 135 AspAspProLysPheArgGlyProPheArgAlaAlaAlaLeuHis 150 GluGlyIleArgAlaCysTrpSerThrProValThrSerGlyAsp 165 GlyThrIleLeuGlyThrPheAlaIleTyrGlySerValProAla 180 PheProAlaGlnGlnAspValAlaLeuValThrGlnCysThrAsp 195 LeuThrAlaAlaValIleThrThrHisLysLeuHisGlnAspLeu 210 SerMetSerGluGluArgPheArgArgAlaPheAspSerAsnVal 225 ValGlyMetAlaLeuLeuAspGluSerGlySerSerIleArgVal 240 AsnAspThrLeuCysAlaLeuThrAlaAlaProProArgArgLeu 255 LeuGlyHisProMetGlnGluIleLeuThrAlaAspSerArgGlu 270 ProPheAlaAsnGlnLeuSerSerIleArgGluGlyLeuThrAsp 285 GlyGlyGlnLeuAspGlyArgIleGlnThrThrGlyGlyArgTrp 300 IleP roValHisLeuSerIleSerGlyMetTrpThrThrGluArg 315 GluPheMetGlyPheSerValHisValLeuAspIleSerGluArg 330 LeuAlaAlaGluArgAlaArgGluGluGlnLeuGluAlaGluVal 345 AlaArgHisThrAlaGluGluAlaSerArgAlaLysSerThrPhe 360 LeuSerGlyMetThrHisGluValGlnThrProMetAlaValIle 375 ValGlyPheSerGluLeuLeuGluThrLeuAspLeuAspGluGlu 390 ArgArgGlnCysAlaTyrArgLysIleGlyGluAlaAlaLysHis 405 ValIleSerLeuValAspAspValLeuAspIleAlaLysIleGlu 420 AlaGlyAlaIleThrLeuGlnAspGluAspIleAspLeuSerGlu 435 GluValAlaThrIleValGluMetLeuGluProIleAlaArgAsp 450 ArgAspArgAspValCysLeuArgTyrValProProGlnThrPro 465 ValHisValCysSerAspArgArgArgValArgGluValLeuLeu 480 AsnIleValSerAsnGlyIleLysTyrAsnArgLeuGlyGlyVal 495 ValAspProProThrGlySerGlyAlaAlaArgProArgGlnThr 510 ArgAlaProAspTyrProAlaThrProThrThrAsnSerSerSer 525 ProSerThrGlyTrpGluSerArgProArgGlyCysLysGlyArg 534 GlySerValLeuArgSerProAlaArg

SEQ ID NO: 3 Sequence length: 735 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Origin Biologic name: Rhodococcus erythropolis
Erythropolis) Strain name: SK92 Sequence: ATG GCC GGA GCG GAC GTC CAC GCC CAG GGT GGC ACG AAT CGA CGT 45 GCA CGC ATC CTC GTC GTC GAC GAC GAA AAA CAC GTG CGC ACG ATG 90 GTG ACG TGG CAA CTC GAA TCG GAG AAT TTC GAT GTT GTC GCT GCG 135 GCA GAC GGA GAT GCG GCA CTG CGT CAG GTC ACT GAG AGC GCA CCC 180 GAT TTG ATG GTG CTC GAT CTG TCG CTC CCG GGG AAA GGT GGG TTG 225 GAA GTG CTC GCT ACG GTC CGC AGA ACC GAT GCA CTG CCT ATC GTC 270 GTG CTC ACA GCA CGC CGC GAT GAA ACC GAA CGG ATC GTC GCG CTG 315 GAT CTC GGC GCC GAT GAC TAC GTC ATC AAA CCG TTC TCC CCG CGG 360 GAA TTG GCC GCC CGT ATC CGG GCA GTG CTT CGT CGA ACC ACA GCT 405 GAA CCC CCA CAC GAG GCG GCG GTT CAG CGA TTC GGT GAC CTA GAG 450 ATC GAC ACC GCT GCG CGC GAG GTT CGG CTC CAC GGG ATA CCG CTC 495 GAG TTC ACC ACC AAG GAG TTC GAT CTG CTG GCC TAT ATG GCC GCA GCA GCA TCA CCG ATG CAG GTC TTC AGC CGA CGC AGA TTG TTG CTC GAG GTG 585 TGG CGA TCG TCG CCC GAC TGG CAG CAG GAC GCC ACC GTG ACC GAG 630 CAC GTG CAC CGC ATT CGC CGC AAG ATC GAA GAA GAT CCC ACC AA A 675 CCG ACG ATC CTG CAG ACA GTG CGG GGA GCC GGT TAC CGT TTC GAC 720 GGA GAG CGT GCA TGA 735

SEQ ID NO: 4 Sequence length: 1605 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Origin Biologic name: Rhodococcus erythropolis
Erythropolis) Strain name: SK92 Sequence: ATG ATG ACC GAC ACA CTG CCC TCC TCG TCC CGT TGG ACC CTT GAA 45 GGC CCG CAT CTC CAG CCG CTG CAG GGT GAG GCC CTG GCG GAT CTC 90 CAC GCC CGT ACG CTC GAG ATG ATC ACT TCC GGG AGA GAA TTG CAC 135 GAG ACA CTC GAG GTG GTC GCC CGC GGC ATC GAG GAA CTG ATG CCG 180 GGC AAA CGT TGC GCA ATT CTG TTG CTC GAC AAC ACC GGA CCG GTA 225 TTG CGC TGC GGC GCG GCC CCA ACA ATG AGC GCG CCG TGG CGC CGG 270 TGG ATC GAC AGC CTC GTC CCT GGT CCG ATG TCG GGT GGC TGC GGC 315 ACA GCG GTT CAC CTC GGC GAG CCG GTT ATT TCC TAT GAC GTG GCC 360 GAT GAC CCG AAA TTC CGC GGC CCC TTC CGC GCC GCA GCC CCC CAC 405 GAG GGC ATA CGT GCC TGC TGG TCC ACC CCC GTC ACA AGC GGA GAC 450 GGC ACG ATC CTC GGC ACT TTC GCG ATC TAC GGA TCC GTG CCG GCG 495 TTC CCC GCA CAA CAG GAC GTT GCC CTG GTC ACC CAA TGC ACC GAC GTC CTG ACC GCT GCC GTC ATC ACC ACC CAC AAA CTT CAT CAA GAT CTG 585 AGC ATG AGC GAG GAG CGG TTC CGA CGC GCC TTC GAT TCC AAT GTC 630 GTC GGC ATG GCA CTT CTC GAC GAA TCC GGC TCC AGC ATC CGC GT C 675 AAC GAC ACC CTG TGC GCG TTG ACC GCA GCT CCG CCA CGG CGC CTC 720 CTC GGC CAC CCC ATG CAG GAG ATA CTC ACC GCC GAC TCC CGG GAA 765 CCG TTC GCC AAT CAG TTG TCC TCC ATC CGT GAG GGA TTG ACC GAC GGC GGA CAG CTC GAC GGA CGA ATC CAA ACC ACC GGA GGT CGG TGG 855 ATT CCG GTG CAC CTG TCC ATC AGC GGT ATG TGG ACC ACG GAG CGG 900 GAG TTC ATG GGA TTC AGC GTC CAT GTC CTG GAC ATC TCC GAG CGC 945 CTG 945 CTG GCC GAA CGC GCC CGC GAG GAA CAA CTC GAG GCC GAG GTT 990 GCC CGC CAT ACC GCG GAG GAA GCC AGT CGC GCC AAG TCC ACG TTC 1035 CTG TCC GGC ATG ACG CAC GAG GTC CAA ACG CCC ATG GCC GTT ATC 1080 GTC GGA TTC GAG CTA CTC GAG ACG CTG GAC CTG GAT GAA GAA 1125 CGT CGT CAG TGC GCC TAC CGC AAG ATC GGC GAA GCC GCG AAA CAC 1170 GTG ATC TCC CTG GTC GAC GAC GTT CTC GAT ATA GCC AAG ATC GAA 1215 GCC GGC GCT ATC ACT CAG GAC GAA GAC ATC GAC CTG TCC GAA 1260 GAA GTT GCC ACC ATC GTG GAG ATG CTC GAG CCC ATC GCC CGT GAC 1305 CGT GAC CGT GAC GTC TGC CTG CGG TAC GTC CCG CCG CAG ACA CCG 1350 GTG CAC GTG TGC TCG GACCG G CGG CGG GTG CGG GAA GTG CTG CTC 1395 AAC ATC GTC TCC AAC GGG ATC AAG TAC AAT CGG CTC GGT GGT GTC 1440 GTC GAC CCC CCA ACA GGA TCA GGG GCT GCT CGT CCG CGT CAG ACG 1485 AGG GCC CCG GAC TAC CCA GCG A CCG ACG ACG AAC TCT TCG AGC 1530 CCT TCA ACC GGC TGG GAG TCG AGG CCA CGG GGG TGC AAG GGT CGG 1575 GGC TCG GTC TTG CGC TCT CCC GCG CGC TGA 1605

SEQ ID NO: 5 Sequence length: 2336 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Origin Biological name: Rhodococcus erythropolis
erythropolis) strain name: SK92 sequence: ATGGCCGGAG CGGACGTCCA CGCCCAGGGT GGCACGAATC GACGTGCACG 50 CATCCTCGTC GTCGACGACG AAAAACACGT GCGCACGATG GTGACGTGGC 100 AACTCGAATC GGAGAATTTC GATGTTGTCG CTGCGGCAGA CGGAGATGCG 150 GCACTGCGTC AGGTCACTGA GAGCGCACCC GATTTGATGG TGCTCGATCT 200 GTCGCTCCCG GGGAAAGGTG GGTTGGAAGT GCTCGCTACG GTCCGCAGAA 250 CCGATGCACT GCCTATCGTC GTGCTCACAG CACGCCGCGA TGAAACCGAA 300 CGGATCGTCG CGCTGGATCT CGGCGCCGAT GACTACGTCA TCAAACCGTT 350 CTCCCCGCGG GAATTGGCCG CCCGTATCCG GGCAGTGCTT CGTCGAACCA 400 CAGCTGAACC CCCACACGAG GCGGCGGTTC AGCGATTCGG TGACCTAGAG 450 ATCGACACCG CTGCGCGCGA GGTTCGGCTC CACGGGATAC CGCTCGAGTT 500 CACCACCAAG GAGTTCGATC TGCTGGCCTA TATGGCCGCA TCACCGATGC 550 AGGTCTTCAG CCGACGCAGA TTGTTGCTCG AGGTGTGGCG ATCGTCGCCC 600 GACTGGCAGC AGGACGCCAC CGTGACCGAG CACGTGCACC GCATTCGCCG 650 CAAGATCGAA GAAGATCCCA CCAAACCGAC GATCCTGCAG ACAGTGCGGG 700 GAGCCGGTTA CCGTTTCGAC GGAGAGCGTG CATGATGACC GACACACTGC 750 CCTCCTCGTC CCGTTGGACC CTTGAAGGCC CGCATCTCCA GCCGCTGCAG 800 GGTGAGGCC C TGGCGGATCT CCACGCCCGT ACGCTCGAGA TGATCACTTC 850 CGGGAGAGAA TTGCACGAGA CACTCGAGGT GGTCGCCCGC GGCATCGAGG 900 AACTGATGCC GGGCAAACGT TGCGCAATTC TGTTGCTCGA CAACACCGGA 950 CCGGTATTGC GCTGCGGCGC GGCCCCAACA ATGAGCGCGC CGTGGCGCCG 1000 GTGGATCGAC AGCCTCGTCC CTGGTCCGAT GTCGGGTGGC TGCGGCACAG 1050 CGGTTCACCT CGGCGAGCCG GTTATTTCCT ATGACGTGGC CGATGACCCG 1100 AAATTCCGCG GCCCCTTCCG CGCCGCAGCC CTCCACGAGG GCATACGTGC 1150 CTGCTGGTCC ACCCCCGTCA CAAGCGGAGA CGGCACGATC CTCGGCACTT 1200 TCGCGATCTA CGGATCCGTG CCGGCGTTCC CCGCACAACA GGACGTTGCC 1250 CTGGTCACCC AATGCACCGA CCTGACCGCT GCCGTCATCA CCACCCACAA 1300 ACTTCATCAA GATCTGAGCA TGAGCGAGGA GCGGTTCCGA CGCGCCTTCG 1350 ATTCCAATGT CGTCGGCATG GCACTTCTCG ACGAATCCGG CTCCAGCATC 1400 CGCGTCAACG ACACCCTGTG CGCGTTGACC GCAGCTCCGC CACGGCGCCT 1450 CCTCGGCCAC CCCATGCAGG AGATACTCAC CGCCGACTCC CGGGAACCGT 1500 TCGCCAATCA GTTGTCCTCC ATCCGTGAGG GATTGACCGA CGGCGGACAG 1550 CTCGACGGAC GAATCCAAAC CACCGGAGGT CGGTGGATTC CGGTGCACCT 1600 GTCCATCAGC GGTATGTGGA CCACGGAGCG GGAGTTCATG GGATTCAG CG 1650 TCCATGTCCT GGACATCTCC GAGCGCCTGG CCGCCGAACG CGCCCGCGAG 1700 GAACAACTCG AGGCCGAGGT TGCCCGCCAT ACCGCGGAGG AAGCCAGTCG 1750 CGCCAAGTCC ACGTTCCTGT CCGGCATGAC GCACGAGGTC CAAACGCCCA 1800 TGGCCGTTAT CGTCGGATTC AGTGAGCTAC TCGAGACGCT GGACCTGGAT 1850 GAAGAACGTC GTCAGTGCGC CTACCGCAAG ATCGGCGAAG CCGCGAAACA 1900 CGTGATCTCC CTGGTCGACG ACGTTCTCGA TATAGCCAAG ATCGAAGCCG 1950 GCGCTATCAC TCTGCAGGAC GAAGACATCG ACCTGTCCGA AGAAGTTGCC 2000 ACCATCGTGG AGATGCTCGA GCCCATCGCC CGTGACCGTG ACCGTGACGT 2050 CTGCCTGCGG TACGTCCCGC CGCAGACACC GGTGCACGTG TGCTCGGACC 2100 GGCGGCGGGT GCGGGAAGTG CTGCTCAACA TCGTCTCCAA CGGGATCAAG 2150 TACAATCGGC TCGGTGGTGT CGTCGACCCC CCAACAGGAT CAGGGGCTGC 2200 TCGTCCGCGT CAGACGAGGG CCCCGGACTA CCCAGCGACG CCGACGACGA 2250 ACTCTTCGAG CCCTTCAACC GGCTGGGAGT CGAGGCCACG GGGGTGCAAG 2300 GGTCGGGGCT CGGTCTTGCG CTCTCCCGCG CGCTGA 2336

[0040]

[Brief description of drawings]

FIG. 1 is a schematic diagram of a deletion plasmid. Arrows on the SK92 strain DNA fragment indicate the position and orientation of the gene encoding the regulatory factor found in the present invention and the nitrilase gene.

FIG. 2 Restriction enzyme map of recombinant plasmid pSK108

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication C12R 1:01) (C12N 9/78 C12R 1:01)

Claims (6)

[Claims] 1. A regulatory factor having an action of activating a nitrilase gene promoter and comprising two components of a polypeptide having the amino acid sequence of SEQ ID NO: 1 and a polypeptide having the amino acid sequence of SEQ ID NO: 2 and the same. Encoding gene DNA. 2. The regulatory factor and gene DNA according to claim 1, wherein the gene encoding the polypeptide has the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4. 3. The regulatory factor and gene DN according to claim 1 or 2, wherein the presence of nitriles enhances the action of activating the nitrilase gene promoter.
A. 4. A recombinant plasmid containing a DNA encoding the regulatory factor according to claim 1, 2 or 3, a nitrilase gene containing a promoter region, and a DNA region capable of replicative growth in a bacterial cell belonging to the genus Rhodococcus. 5. A DNA region capable of growing in a bacterial cell belonging to the genus Rhodococcus has plasmids pRC001 and pRC.
The recombinant plasmid according to claim 4, wherein the recombinant plasmid is selected from 002, pRC003 or pRC004. 6. A microorganism belonging to the genus Rhodococcus transformed with the recombinant plasmid according to claim 4 or 5.